351 Practical II Differential Tests Review

Ex. 5-2Phenol Red (PR)- Fermentation glucose, sucrose,

lactose for Escherichia coli

• Lac (left) gas+• Glu( middle) gas + • Suc (right) no gas –

• Phenol red indicator used to see if fermentation has occurred. Durham tubes are red before any fermentation has occurred. Fermentation produces gas and/or acid from the breadkdown of carbohydrates

Ex. 5-2Phenol Red (PR) Fermentation glucose, sucrose, lactose for Alcaligenes faecalis

• Suc (left) –• Lac (middle) –• Glu (right) –

• Think about why A. faecaliscould not breakdown glu,suc, or lac?

This is a negative This is a negative result, must have result, must have full yellow to be full yellow to be positive. Donpositive. Don’’t t worry the exam worry the exam ones will be more ones will be more obvious obvious ☺☺!!

Ex. 5-2Phenol Red (PR) Fermentation glucose,

sucrose, lactose for Saccharomyces cerevisiae

• Lac (left) –• Glu (middle) gas• Suc (right) –

Why did S. cerevisiaeNOT change color?

Ex. 5Ex. 5--44Methyl Red (MR) (IMViC tests)

• Enterobacter aerogenes (left) –

• E. coli (bright red) +

• Reagent: Methyl red indicator identifies pH change due to mixed acid fermentation

Ex. 5Ex. 5--44Voges – Proskauer (VP)

(IMViC tests)• Enterobacter aerogenes +

• E. coli – (left)

• Barritt’s reagent Tests for acetoin, precursor to 2,3 butanediol fermentation

• Addition of alpha-naptholand KOH

This is the beginning of the reaction, you should see a cherry red color throughout inoculation!

Ex. 5Ex. 5--55Catalase

• Bubbles +• No bubbles –• Reagents 3% H2O2Tests for the ability to break down toxic O 2 products/superoxide

dismutase (catalyzes the destruction of superoxide) & catalase operoxidase (catalyzes the destruction of hydrogen peroxide)

2 O2-+ 2 H+ ---superstable dismutate O 2 + H2O2

2 H2O2 ---catalase 2 H2O + O2

Ex. 5Ex. 5--66Oxidase

• Blue (30 sec) +• No color change –• Tests done on Oxidase strips• Tests for the oxidation of reduced cytochrome c to form

water and reduced cytochrome c / Cytochrome oxidase

Oxidized cyt C + reagent Wurster’s blue + red cyt C

clear dark purpleoxidized

Ex. 5Ex. 5--77Nitrate

• Red color after reagents/no color after zinc + Escherichia coli (right)

• No color change after zinc is a + for denitrification to nitrogen gas or ammonia

Soil- (not pictured, would have a gas bubble in durham tube)

• Color change after Zn added will be –for nitrate reductaseMicrococcus luteus (left)Alcaligenes faecalis (middle)

• Reduction of nitrate to nitrite to be used as a final electron acceptor/Nitrate reductase

Ex. 5Ex. 5--88Citrate (IMViC tests)

• E. coli (left green) –

• Enterobacter aerogenes (right royal blue) +

• Reagent: Bromothymol blue indicator tests for ability to use citrate as sole carbon source/citrate permease

Ex. 5Ex. 5--1313Starch hydrolysis

• Zone of clearing +• No zone –• Bacillus subtillis +,

Alcaligenes faecalis –Escherichia coli – (Clockwise)

• Iodine must be on the plate to visualize the zone of clearing surrounding the bacteria. This zone indicates starch was broken down to dextrins, maltose, and glucose/alpha-amylase

Ex. 5Ex. 5--1515Urease

Phenol Red a pH indicator turns tube bright pink because NH3 decreases the pH

CO(NH3)2 + 2 H2O –urease CO2+ H2O + 2 NH3

E. coli – (left)Proteus vulgaris +

Ex. 5Ex. 5--1616Casein hydrolysis

• Zone of clearing +• No zone –• Test used to see If

casein is degraded into amino acids for use as a carbon source/proteolytic enzymes

• Escherichia coli – , Alcaligenes faecalis –Bacillus subtilis +

Ex. 5Ex. 5--1717Gelatin hydrolysis

• Liquid on gelatin +• No liquid –• Hydrolysis of gelatin

into amino acids to be used as nutrients/gelatinase

• Escherichia coli (top) –• Bacillus spp. +

Ex. 5Ex. 5--1919Lipid Hydrolysis

For the Egg Yolk agar, the growth must have a white halo around the colony growth if it utilizes the lipids therefore having the enzyme lipase (hard to see in pics!). Bacillus spp. +Escherichia coli –Alcaligenes faecalis –

Ex. 5Ex. 5--2020Sulfur reduction test, Indole production, Motility

(SIM) deepsall 3 tests done w/SIM deeps just add Kovac’s reagent for Indole test

• Alcaligenes faecalis (left) -• Escherichia coli (middle) –• Proteus vulgaris (black

precipitate) +

• Reagent: Ferrous ammonium sulfate-indicator. H2S reacts w/ ferrous sulfate forming the black precipitate Sodium thiosulfate is reduced to sulfite/thiosulfate

Indole (IMViC tests)

• Enterobacter aerogenes –• Escherichia coli (pink/red) +• Kovac’s reagent detects if

tryptophan has been hydrolyzed to indol/tryptophanase

Ex. 5Ex. 5--2323Litmus Milk

Ex. 5Ex. 5--2424Bacitracin

Susceptibility

From Left to Right:

1. Control (NC)

2. Enterococcus faecalis (R)

3. Bacillus megaterium (D)

4. Proteus vulgaris (C)5. Alcaligenes faecalis (K)6. Latocococcus lactis (AC)7. Escherichia coli (A)

**See Page 187 in book for classifications

Ex. 5Ex. 5--2626Blood Agar: Hemolysis

• Check which bacteria are capable of lysing red blood cells (RBCs) by using blood agar (sheep blood).

• α = partial lysis of red blood cells blood looks greenish

• β = complete lysis of blood clearing

• γ = no lysing• Clockwise starting from the left:

Staphylococcus aureus β, Staphylococcus epidermidis γ , teeth α

Ex. 5Ex. 5--2727Coagulase

• Results:+ clotting in thebottom of the broth • Reagents:Plasma• Reason/Enzymes Clots plasma to avoid attack by

host’s defenses/Coagulase

Staphylococcus aureus +; Staphylococcus epidermidis –

Ex. 5Ex. 5--2828Motility

• Spreading growth +(Spreading growth looks like a

mascara brush in the deep)Escherichia coli (right)Proteus vulgaris (left)

• Linear growth –Staphylococcus epidermidis(middle)

• To test for the ability of bacterium to migrate in solid agar deep

Ex. 7Ex. 7--33Antibiotic

• Ability of antibiotics to inhibit growth on Mueller-Hinton agar plates (Whether bacteria are susceptible, intermediate, or resistant depends on the amount of antibiotic and the diameter of zone of inhibition, check table 43.1 of your lab manual )

Mannitol salt• Mannitol salt agar is a

selective and differential medium used for differentiating between different stapylococci

• Staphylococcus aureuschanges medium to yellow

• Staphylococcus epidermidiswill not change the medium

• Why does S. aureus change the color of this medium?

About…351 Practical II Review Slides

• These slides are manufactured by students, if you see some error, please contact me at fester@unlv.nevada.edu

• Most of these slides were contributed by Austin McDonald from the 351 Fall 2007 Class. Thanks Austin!!

mailto:fester@unlv.nevada.edu

351 Practical II Differential Tests ReviewEx. 5-2 Phenol Red (PR)- Fermentation glucose, sucrose, lactose for Escherichia coliEx. 5-2Phenol Red (PR) Fermentation glucose, sucrose, lactose for Alcaligenes faecalisEx. 5-2Phenol Red (PR) Fermentation glucose, sucrose, lactose for Saccharomyces cerevisiaeEx. 5-4Methyl Red (MR) (IMViC tests)Ex. 5-4Voges – Proskauer (VP) (IMViC tests)Ex. 5-5CatalaseEx. 5-6OxidaseEx. 5-7 NitrateEx. 5-8Citrate (IMViC tests)Ex. 5-13Starch hydrolysisEx. 5-15UreaseEx. 5-16Casein hydrolysisEx. 5-17Gelatin hydrolysisEx. 5-19Lipid HydrolysisEx. 5-20Sulfur reduction test, Indole production, Motility (SIM) deepsall 3 tests done w/SIM deeps just add Kovac’s reagentIndole (IMViC tests)Ex. 5-24 Bacitracin SusceptibilityEx. 5-26Blood Agar: HemolysisEx. 5-27CoagulaseEx. 5-28MotilityEx. 7-3AntibioticMannitol saltAbout…351 Practical II Review Slides